Studies on drug resistance in HIV-related Kaposi sarcoma

卡波西肉瘤（KS）是一种由人类疱疹病毒8引起的肿瘤，通常发生在免疫系统较弱的艾滋病患者身上。在艾滋病相关的KS病例中，高活性抗逆转录病毒治疗（HAART）可诱导疾病显著消退；然而，HAART并不总是有效的，二线治疗包括化疗干预。蒽环类药物，如阿霉素，经常用于治疗艾滋病相关的KS，但对这些药物的耐药性确实出现了。紫杉醇和多西紫杉醇等紫杉醇类药物已成功用于治疗阿霉素耐药KS。然而，尚无研究对紫杉烷的潜在耐药机制进行研究。由于在治疗艾滋病相关的KS时，HAART通常与化疗同时进行，因此检查最近批准的抗病毒药物对用于治疗KS的化疗的潜在耐药机制的影响是很重要的。我们对卡波西肉瘤耐药机制的兴趣来自于我们之前对atp依赖性转运蛋白p -糖蛋白（P-gp）的研究。我们之前证明了P-gp可以抑制HIV病毒的产生，并降低过表达这种转运体的细胞的传染性。此外，许多HIV蛋白酶抑制剂已被证明是P-gp的底物。在一种卡波西肉瘤细胞系模型中，我们发现蒽环类药物治疗导致P-gp表达增加，而HIV蛋白酶抑制剂可以进一步增强这种表达。然而，用于这项研究的细胞系最近被证明是错误的，因此需要重新评估这些结果。我们目前的工作重点是L1T2细胞系，该细胞系已被证明在小鼠模型中概括了卡波西肉瘤的临床特征。我们鉴定了该细胞系对紫杉醇和HIV蛋白酶抑制剂的耐药模式，并分离了L1T2细胞系的耐药亚系，利用Taq-man低密度阵列微流控芯片检测380个耐药相关基因的mRNA表达，这些基因包括涉及药物外排、DNA修复机制、细胞凋亡和代谢酶的基因。FACS分析和Western Blot分析显示，在紫杉醇和奈非那韦（蛋白酶抑制剂）选择的细胞中，ABCB1 （p -糖蛋白）的表达增加。药物转运体活性测定显示，在奈非那韦或紫杉醇选择的细胞中，由于P-gp转运体活性较高，罗丹明123的外排率较高。根据这些结果，长期使用奈非那韦的细胞对紫杉醇表现出更高的耐药性，而添加P-gp抑制剂缬斯帕达使这些细胞对药物更敏感。这些结果提示HIV蛋白酶抑制剂可能在诱导化疗耐药中起作用。最终，我们希望对卡波西肉瘤耐药新机制的鉴定将导致治疗方法的改进。

Kaposi's Sarcoma (KS) is a type of tumor caused by the human herpesvirus 8 that often develops in AIDS patients with weakened immune systems. In the case of AIDS-related KS, highly active antiretroviral therapy (HAART) can induce significant regression of the disease; however, HAART is not always effective and second-line therapies for KS include chemotherapeutic intervention. Anthracyclines such as doxorubicin are frequently used in the treatment of AIDS-related KS but resistance to these agents does arise. Taxanes such as paclitaxel and docetaxel have successfully been used to treat doxorubicin-resistant KS. However, no studies have examined the potential resistance mechanisms to taxanes in KS. As HAART is often administered concomitantly with chemotherapy when treating AIDS-related KS, it is important to examine the effects of recently-approved antivirals on potential resistance mechanisms to chemotherapy used to treat KS. Our interest in resistance mechanisms in Kaposi's sarcoma comes from our previous work with the ATP-dependent transporter P-glycoprotein (P-gp). We previously demonstrated that P-gp can hamper HIV virus production and decrease infectivity in cells that overexpress the transporter. Additionally, a number of HIV protease inhibitors have been shown to be substrates of P-gp. In one cell line model of Kaposi's sarcoma, we found that anthracycline treatment leads to increased P-gp expression and that HIV protease inhibitors can further augment this expression. However, the cell line used for this study was recently shown to be mischaracterized, thus requiring a reevaluation of these results. Our current work focuses on the L1T2 cell line that has been shown to recapitulate clinical features of Kaposi's sarcoma in mouse models. We have characterized the drug resistance pattern of this cell line selected for resistance to paclitaxel and HIV protease inhibitors and have isolated resistant sublines of the L1T2 line to identify potential mechanisms of resistance using a Taq-man low-density array microfluidic chip to detect mRNA expression of 380 drug resistance-related genes comprising genes involved in drug efflux, DNA repair mechanisms, apoptosis and metabolizing enzymes. FACS analysis as well as Western Blot analysis show increased expression of ABCB1 (P-glycoprotein) in both paclitaxel and nelfinavir (protease inhibitor)-selected cells. Drug transporter activity assays show higher efflux of Rhodamine123 in cells selected with nelfinavir or taxol due to higher P-gp transporter activity. In accordance with those results, cells under prolonged selection with Nelfinavir showed higher resistance to taxol, whereas addition of valspadar, a P-gp inhibitor, made these cells more sensitive to the drug. These results suggest that HIV protease inhibitors may play a role in inducing resistance to chemotherapy. Ultimately we hope that identification of novel mechanisms of resistance in Kaposi's sarcoma will lead to improved therapies.